| IntroductionGastric carcinoma is one of the most common malignant tumor in the world, and the most popular malignant tumor in China. It was demonstrated that the carcinogenesis of gastric carcinoma is a multi - step process participated with polygene.Tumor suppressor gene (TSG) is one of negative regulation signals on cell proliferation, differentiation and apoptosis process, and one of hot spots in oncological study. Regulation system of cell cycle is essential to sustain normal cell cycles. Furthermore, disorders of regulation can be caused by the abnormal of key factors in this regulation system, and will result in the abnormal of cell proliferation, unable to differentiate and apoptosis promptly and occurrence of tumor.It has been popular presumed that abnormal methylation is the third mechanism except absence and mutation which would result in inactivation of TSGs, and plays an important role in tumorigenesis and development. Methylation is a natural modification style of DNA. This basic group modification process is catalyzed by DNA methyltransferase (DNMT) which would add a methyl (CH3 - ) to DNA. Most of methylated cytosine in human and animals located in CpG islands, and CpG in human genome is only account for 10%. Among them, 70% to 80% are in methylation situation. The methylation process of gene is a suppressive procedure of genetic expression. What is more, both methylation of TSGs and promoters of gene are closely related with tumorigenesis.p15 gene and p16 gene are cyclin dependence kinase inhibitors (CDKI)that closely related to tumorigenesis and suppress cell proliferation. Inactivation of them will help absoluteness proliferation of tumor cells and promote tumor formation. Many studies found that methylation in promoter region of pl6 gene was related to leukemia, gastric carcinoma, hepatic carcinoma, colorectal carcinoma, esophageal carcinoma and so on. It will be helpful to reveal the regulation of cell cycle and provide clues to elucidate process of cell proliferation, differentiation, and cancerization with the study on this aspect. Furthermore, it will probably provide theoretical and experimental evidences to clinical diagnosis, treatment and prognosis of tumors.Methylation - specific PCR ( MSP) and immunohistochemistry methods were used in this experiment to detect methylation level and protein expression of pl5 gene and pl6 gene in primary focus, metastasis and adjuvant non - cancerous tissue of gastric carcinoma that aim at the investigation of relationship between methylation of pl5 gene and pl6 gene and pathobiological behavior of metastasis of gastric carcinoma.MethodsFifty cases of matched paraffin embedded tissues from resected primary focus , metastasis and adjuvant non - cancerous tissue of gastric carcinoma used in this experiment were collected from gastric carcinoma patients treated in gastric surgical department of Cancer Hospital of Iiaoning Province from September 1998 to March 1999. Among them, thirty - two were male and eighteen were female. The male female ratio ( M - F ratio) was 1.78 vs 1 and mean age was 57. 11 ±8.61 (79 to 39). Carcinomas were staged according to TNM stage system of UICC and typed under Japanese gastric carcinoma management protocol. Adjuvant non - cancerous tissues were non - cancerous epithelium mucosae. Both kinds of tissues were confirmed by pathologic diagnosis and no pre - operative chemotherapy and radiotherapy were performed on these patients. Methylation level on promoter region of pl5 gene and pl6 gene were detected with MSP and validated by detecting pi5 and pl6 protein with immnohistochemistry method. Chi - squared test or Fisher's exact propability test and non - parameter rank -sum test were used in comparison of ratio between groups. Experimental results were analyzed with the use of statistical package of SPSS (version 10. 0) , and differences were considered statistically significant when p value less than 0. 05 as size of statistical test was a =0.05.Results1. Positive rate of methylation on promoter region of pi5 gene was 62.0% (31/50) in 50 cases of gastric carcinoma, 86. 8% (46/53) in metastasis and 6.0% (3/50) in corresponding non -cancerous tissues. Methylation levels in cancerous tissues, metastasis were higher than that of non - cancerous tissues with statistical significant (P < 0. 01) .2. Positive rate of methylation on promoter region of pl6 gene was 66.0% (33/50) in 50 cases of gastric carcinoma, 96.2% (51/53) in metastasis and 8.0% (4/50) in corresponding non -cancerous tissues. Methylation levels in cancerous tissues, metastasis were higher than that of non - cancerous tissues with statistical significant ( P < 0. 01) .3. Fifty patients were divided into differentiated type group and undifferen-tiated type group according to differentiation degree of cancerous tissues. Both of positive rates of methylation on promoter region of pl5 gene and pl6 gene in un-differentiated type group were 40. 9% (9/22). They were lower than those of 78. 6% (22/28) and 85.7% (24/28) in differentiated type group with statistical significant ( P < 0. 05 ). Positive rates of methylation on promoter region of pi5 gene in metastasis were different between differentiated type of 70. 8% (17/ 24) and undifferentiated type of 100% (29/29) with statistical significant (P < 0.01). However, no difference with statistic significant was found between methylation positive levels on promoter region of pl6 gene in metastasis. Those were 91.7% (22/24) in differentiated type and 100% (29/29) in undifferentiated type (P>0.05).4. All tumors were divided into Borrmann I, II, III and IV type according to their general type, and methylation positive rates step up gradually among different groups with statistical significance ( P <0.05). 5. Fifty tumors were di-vided into massive type, nest type and infiltrating type, and methylation positive rate on promoter region of pl5 gene and pl6 gene in primary focus and that of pi5 gene in metastasis were step up gradually with statistical significance (P < 0.01). However, no statistical significance was found among methylation positive rates on promoter region of pi6 gene between three growth patterns in metastasis (P>0.05).6. Fifty tumors were divided into Tl, T2, T3, and T4 groups according infiltrating depth, and methylation positive rate on promoter region of pi5 gene and pi 6 gene in primary focus and that of pi 5 gene in metastasis were step up gradually with statistical significance (P <0. 01). However, no statistical significance was found among methylation positive rates on promoter region of pi 6 gene between four groups in metastasis (P >0.05).7. Fifty patients were divided into four groups according to their survival time, as less than one year, less than three years, less than five years and more than five years, methylation positive rate on promoter region of pi5 gene and pi6 gene in primary focus of differentiated type and that of pi5 gene in metastasis were step down gradually with statistical significance ( P <0.01) . However, no statistical significance was found among methylation positive rates on promoter region of pi6 gene between four groups in metastasis (P >0.05).Conclusion1. Methylations on promoter region of pi5 and pi6 genes are probably important molecule events during cancerization of gastric carcinoma.2. There are correlation between the protein of TSGs, pl5 and pl6 and methylations on promoter region of TSGs, pi5 and pi6.3. Methylation level on promoter region of pl5 and pl6 genes correlate with biological behaviours of gastric carcinoma, and was higher in tumors with high malignant degree.4. Correlation between survival time of gastric carcinoma patients and methylations on promoter region of pi5 and pi6 genes was found, and could be used as one of prognostic indicators. But, no relationship between survival time...
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